Free Radical–Initiated Peptide Sequencing Mass Spectrometry for Phosphopeptide Post-translational Modification Analysis

Jang, Inae; Jeon, Aeran; Lim, Suk Gyu; Hong, Duk Ki; Kim, Min Soo; Jo, Jae Hyeong; Lee, Sang Tak; Moon, Bongjin; Oh, Han Bin

doi:10.1007/s13361-018-2100-1

Cited by 9 publications

(9 citation statements)

References 66 publications

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“…Here, it is worthwhile to mention that, in the case of UBT(Gu), twostep collisional activations were not necessary for backbone fragmentations, unlike in the case of TEMPO−Bz−NH− peptide, wherein two-step collisional activations were required in the positive-ion mode. 50,51 Analysis of the FRIPS MS/MS spectra showed that a-/x-and c-/z-type fragments were the main product types in protein backbone fragmentations, whereas b-and y-type fragments were observed as minor products. This observation clearly indicates that the radicaldriven fragmentations are the major fragmentation pathway for low charge state UBT(Gu) ions, even when UBT(Gu) ions are subjected to high collision energy (i.e., NCE > 25).…”

Section: Fragmentation Characteristics Of Ubt(gu)mentioning

confidence: 99%

“…In this study, we report a new approach to characterize the protein structure in the gas phase using TEMPO ((2,2,6,6-tetramethylpiperidine-1-yl)-oxyl)-assisted free-radical-initiated peptide sequencing (FRIPS) MS (see Scheme ). − The TEMPO-assisted FRIPS MS is a radical-based fragmentation technique, in which the radical precursor (i.e., TEMPO–Bz–CO− ) is conjugated to the N-terminus of a peptide ( TEMPO–Bz–CO–NH–peptide ). When thermal activation is given to the peptide precursor, a free radical is generated through homolytic bond cleavage between the benzylic carbon and the oxygen of the TEMPO moiety, leading to the formation of •Bz–CO–NH–peptide.…”

Section: Introductionmentioning

confidence: 99%

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TEMPO-Assisted Free-Radical-Initiated Peptide Sequencing Mass Spectrometry for Ubiquitin Ions: An Insight on the Gas-Phase Conformations

Lee

Park

et al. 2022

J. Am. Soc. Mass Spectrom.

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TEMPO ((2,2,6,6-tetramethylpiperidine-1-yl)oxyl)-assisted free-radical-initiated peptide sequencing mass spectrometry (FRIPS MS) is applied to the top-down tandem mass spectrometry of guanidinated ubiquitin (UB(Gu)) ions, i.e., p-TEMPO–Bn–Sc–guanidinated ubiquitin (UBT(Gu)), to shed a light on gas-phase ubiquitin conformations. Thermal activation of UBT(Gu) ions produced protein backbone fragments of radical character, i.e., a-/x- and c-/z-type fragments. It is in contrast to the collision-induced dissociation (CID) results for UB(Gu), which dominantly showed the specific charge-remote CID fragments of b-/y-type at the C-terminal side of glutamic acid (E) and aspartic acid (D). The transfer of a radical “through space” was mainly observed for the +5 and +6 UBT(Gu) ions. This provides the information about folding/unfolding and structural proximity between the positions of the incipient benzyl radical site and fragmented sites. The analysis of FRIPS MS results for the +5 charge state ubiquitin ions shows that the +5 charge state ubiquitin ions bear a conformational resemblance to the native ubiquitin (X-ray crystallography structure), particularly in the central sequence region, whereas some deviations were observed in the unstable second structure region (β2) close to the N-terminus. The ion mobility spectrometry results also corroborate the FRIPS MS results in terms of their conformations (or structures). The experimental results obtained in this study clearly demonstrate a potential of the TEMPO-assisted FRIPS MS as one of the methods for the elucidation of the overall gas-phase protein structures.

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Section: Fragmentation Characteristics Of Ubt(gu)mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

TEMPO-Assisted Free-Radical-Initiated Peptide Sequencing Mass Spectrometry for Ubiquitin Ions: An Insight on the Gas-Phase Conformations

Lee

Park

et al. 2022

J. Am. Soc. Mass Spectrom.

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“…Chemical labeling has also been a pervasive strategy in BU proteomics methods, including free-radical-initiated peptide sequencing (FRIPS). , In this approach, peptides are tagged by a moiety capable of forming a free radical upon collisional activation. The radical then attacks the peptide backbone producing fragment ions similar to electron capture/transfer methods. , Typically, the FRIPS fragment ions are collected alongside CID fragment ions, enabling the integration of both channels of information for enhanced sequence coverage. , FRIPS fragmentation produces many of the benefits commonly associated with ExD experiments, including PTM characterization and localization, , disulfide bond reduction, , and side-chain cleavages that allow for confirmation of sequence assignments…”

Section: Introductionmentioning

confidence: 99%

Free Radical-Based Sequencing for Native Top-Down Mass Spectrometry

Ramírez

Murtada

Gao

et al. 2022

J. Am. Soc. Mass Spectrom.

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Native top-down proteomics allows for both proteoform identification and high-order structure characterization for cellular protein complexes. Unfortunately, tandem MS-based fragmentation efficiencies for such targets are low due to an increase in analyte ion mass and the low ion charge states that characterize native MS data. Multiple fragmentation methods can be integrated in order to increase protein complex sequence coverage, but this typically requires use of specialized hardware and software. Free-radical-initiated peptide sequencing (FRIPS) enables access to charge-remote and electron-based fragmentation channels within the context of conventional CID experiments. Here, we optimize FRIPS labeling for native top-down sequencing experiments. Our labeling approach is able to access intact complexes with TEMPO-based FRIPS reagents without significant protein denaturation or assembly disruption. By combining CID and FRIPS datasets, we observed sequence coverage improvements as large as 50% for protein complexes ranging from 36 to 106 kDa. Fragment ion production in these experiments was increased by as much as 102%. In general, our results indicate that TEMPObased FRIPS reagents have the potential to dramatically increase sequence coverage obtained in native top-down experiments.

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“…Mobile proton-driven processes are often the lowest energy even-electron processes and have been avoided through the study of anions, sequestration of protons via the guanidation of lysine residues, or focus on ions with fewer protons than arginine residues. 2,7,[9][10][11] To be employed in more complex systems and proteomics workflows, the efficiency of sequence ion generation must be improved for multiply charged ions which are frequently generated in proteomics workflows.…”

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confidence: 99%

Improved Performance of Positive-ion mode Free Radical-Initiated Peptide Sequencing with para-TEMPO-Bz

Osho

Wasik

Geary

et al. 2022

Preprint

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Tandem mass spectrometry (MS/MS) is often employed for the sequencing of protein ions and the annotation of protein post-translational modification. Collision-induced dissociation (CID) and electron transfer dissociation (ETD) are the most frequently utilized MS/MS techniques, but both have significant limitations. CID is often funneled into a few kinet-ically labile bonds limiting the effectiveness of sequencing and ETD, is only applicable to multiply charged ions and re-quires specialized instrumentation. Free radical-initiated peptide sequencing (FRIPS) is an alternative fragmentation technique that generates sequence informative ions via collisionally-initiated radical chemistry. Its main advantages over the previously mentioned fragmentation techniques include charge state independence, improved efficiency of sequence ion generation, and applicability to most MS instrumentation. Collision activation (CA) homolytically cleaves the radical precursor, initiates radical formation, extensive hydrogen atom transfer, and peptide backbone dissociation. Although the FRIPS technique shows great promise when applied to cations, radical initiation competes directly with typical CID path-ways. When multiply charged ions of conjugated ACTH 1-14, b-amyloid 10-20, substance p, and melittin are collisionally activated, a series of high abundance mass losses are observed which syphon ion abundance from radically generated sequence ions. This loss of ion abundance reduces the sequence coverage generated by FRIPS fragmentation. In this work, we hypothesized that these mass losses were instigated by the ortho-orientation of the radical precursor undergoing fac-ile conversion into five- or six-membered intermediates and that the para-precursor would not undergo this chemistry. To test this assertion, we synthesized para-TEMPO-Bz, conjugated it to these peptides, and collisionally activated each. And indeed, we see the complete elimination of these undesired collisional processes and the significant increase in radi-cal precursor ion abundance. Moreover, we see a significant increase in the sequence coverage generated when the p-TEMPO-Bz is utilized. From these results, p-TEMPO-Bz significantly improves the performance of positive-ion mode FRIPS and may be a suitable alternative to the currently utilized ortho-TEMPO-Bz-based FRIPS.

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Free Radical–Initiated Peptide Sequencing Mass Spectrometry for Phosphopeptide Post-translational Modification Analysis

Cited by 9 publications

References 66 publications

TEMPO-Assisted Free-Radical-Initiated Peptide Sequencing Mass Spectrometry for Ubiquitin Ions: An Insight on the Gas-Phase Conformations

TEMPO-Assisted Free-Radical-Initiated Peptide Sequencing Mass Spectrometry for Ubiquitin Ions: An Insight on the Gas-Phase Conformations

Free Radical-Based Sequencing for Native Top-Down Mass Spectrometry

Improved Performance of Positive-ion mode Free Radical-Initiated Peptide Sequencing with para-TEMPO-Bz

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